Probes for INS

Telomerase consists of at least two essential elements, an RNA component hTR or TERC that contains the template for telomere DNA addition, and a catalytic reverse transcriptase (TERT). While expression of TERT has been considered the key rate limiting component for telomerase activity, increasing evidence suggests an important role for the regulation of TERC in telomere maintenance and perhaps other functions in human cancer. By using three orthogonal methods including RNAseq, RT-qPCR, and an analytically validated chromogenic RNA in situ hybridization assay, we report consistent overexpression of TERC in prostate cancer. This overexpression occurs at the precursor stage (e.g. high grade prostatic intraepithelial neoplasia or PIN), and persists throughout all stages of disease progression. Levels of TERC correlate with levels of MYC (a known driver of prostate cancer) in clinical samples and we also show the following: forced reductions of MYC result in decreased TERC levels in 8 cancer cell lines (prostate, lung, breast, and colorectal); forced overexpression of MYC in PCa cell lines, and in the mouse prostate, results in increased TERC levels; human TERC promoter activity is decreased after MYC silencing; and MYC occupies the TERC locus as assessed by chromatin immunoprecipitation (ChIP). Finally, we show that knockdown of TERC by siRNA results in reduced proliferation of prostate cancer cell lines. These studies indicate that TERC is consistently overexpressed in all stages of prostatic adenocarcinoma, and its expression is regulated by MYC. These findings nominate TERC as a novel prostate cancer biomarker and therapeutic target.

Abstract

BACKGROUND:

Loss or mutation of PTEN alleles at 10q23 in combination with 8q24 amplification (encompassing MYC) are common findings in aggressive, human prostate cancer. Our group recently developed a transgenic murine model of prostate cancer involving prostate-specific Pten deletion and forced expression of MYC under the control of the Hoxb13 promoter. MYC overexpression cooperated with Pten loss to recapitulate lethal, human prostate cancer.

METHOD:

We now report on the generation of two mouse prostate cancer cell lines, BMPC1 and BMPC2, derived from a lymph node, and liver metastasis, respectively.

RESULTS:

Both cell lines demonstrate a phenotype consistent with adenocarcinoma and grew under standard tissue culture conditions. Androgen receptor (AR) protein expression is minimal (BMPC1) or absent (BMPC2) consistent with AR loss observed in the BMPC mouse model of invasive adenocarcinoma. Growth in media containing charcoal-stripped serum resulted in an increase in AR mRNA in BMPC1 cells with no effect on protein expression, unless androgens were added, in which case AR protein was stabilized, and showed nuclear localization. AR expression in BMPC2 cells was not effected by growth media or treatment with androgens. Treatment with an anti-androgen/castration or androgen supplemented media did not affect in vitro or in vivo growth of either cell line, irrespective of nuclear AR detection.

DISCUSSION:

These cell lines are a novel model of androgen-insensitive prostatic adenocarcinoma driven by MYC over-expression and Pten loss.

Loss of pancreas β-cell function is the precipitating factor in all forms of diabetes. Cell replacement therapies, such as islet transplantation, remain the best hope for a cure; however, widespread implementation of this method is hampered by availability of donor tissue. Thus, strategies that expand functional β-cell mass are crucial for widespread usage in diabetes cell replacement therapy. Here, we investigate the regulation of the Hippo-target protein, Yes-associated protein (Yap), during development of the endocrine pancreas and its function after reactivation in human cadaveric islets. Our results demonstrate that Yap expression is extinguished at the mRNA level after neurogenin-3-dependent specification of the pancreas endocrine lineage, correlating with proliferation decreases in these cells. Interestingly, when a constitutively active form of Yap was expressed in human cadaver islets robust increases in proliferation were noted within insulin-producing β-cells. Importantly, proliferation in these cells occurs without negatively affecting β-cell differentiation or functional status. Finally, we show that the proproliferative mammalian target of rapamycin pathway is activated after Yap expression, providing at least one explanation for the observed increases in β-cell proliferation. Together, these results provide a foundation for manipulating Yap activity as a novel approach to expand functional islet mass for diabetes regenerative therapy.

Abstract

Objectives

Recent commercialization of methods for in situ hybridization using Z-pair probe/branched DNA amplification has led to increasing adoption of this technology for interrogating RNA expression in formalin-fixed, paraffin-embedded (FFPE) tissues. Current practice for FFPE block storage is to maintain them at room temperature, often for many years.

Methods

To examine the effects of block storage time on FFPE tissues using a number of RNA in situ probes with the Advanced Cellular Diagnostic’s RNAscope assay.

Results

We report marked reductions in signals after 5 years and significant reductions often after 1 year. Furthermore, storing unstained slides cut from recent cases (<1 year old) at –20°C can preserve hybridization signals significantly better than storing the blocks at room temperature and cutting the slides fresh when needed.

Conclusions

We submit that the standard practice of storing FFPE tissue blocks at room temperature should be reevaluated to better preserve RNA for in situ hybridization.

The pituitary gland is a primary endocrine organ that controls major physiological processes. Abnormal development or homeostatic disruptions can lead to human disorders such as hypopituitarism or tumours. Multiple signalling pathways, including WNT, BMP, FGF and SHH regulate pituitary development but the role of the Hippo-YAP1/TAZ cascade is currently unknown. In multiple tissues, the Hippo kinase cascade underlies neoplasias; it influences organ size through the regulation of proliferation and apoptosis, and has roles in determining stem cell potential. We have used a sensitive mRNA in situ hybridisation method (RNAscope) to determine the expression patterns of the Hippo pathway components during mouse pituitary development. We have also carried out immunolocalisation studies to determine when YAP1 and TAZ, the transcriptional effectors of the Hippo pathway, are active. We find that YAP1/TAZ are active in the stem/progenitor cell population throughout development and at postnatal stages, consistent with their role in promoting the stem cell state. Our results demonstrate for the first time the collective expression of major components of the Hippo pathway during normal embryonic and postnatal development of the pituitary gland.